Figure 5. Tara-KD regulates the p38MAPK/Tbx3 pathway downstream of Rac1. (A and B) Immunoblotting for phospho-p38MAPK in Tara-KD cells in the presence or absence of SB203580 (A) or ITX3 (B). The phosphorylation level of p38MAPK was markedly up-regulated in Tara-KD cells compared with control cells. SB203580, a p38MAPK phosphorylation inhibitor, dose dependently decreased the phosphorylation level of p38, as did ITX3, an inhibitor of the Rac1-GEF activity of Trio RhoGEF. The bottom panels show the quantification of the immunoblotting data (as shown in Fig. S4 C). The results are expressed as means ± SD (error bars) and are representative of three independent experiments. **, P < 0.01; *, P < 0.05. Densitometric quantification of Western blot bands was performed using Photoshop 7.0 (Adobe). As to the “Relative Intensity,” the ratio of intensities of P-p38 to p38 in control was normalized to 1.0. (C) Immunoblotting for E-cadherin and cadherin-6 in Tara-KD cells. The Tara-KD–induced decrease of E-cadherin and increase of cahderin-6 were dose dependently inhibited by the presence of SB203580. The bottom panels show the quantification of the immunoblotting data (Fig. S4 C). The basically same results were obtained with another inhibitor of p38, SB202190. The results are expressed as means ± SD (error bars) and are representative of three independent experiments, in which antigens were immunoblotted in the same membranes. **, P < 0.01; *, P < 0.05. Densitometric quantification of Western blot bands was performed using Photoshop 7.0 (Adobe). As to the “Relative Intensity,” the ratio of intensities of E-cadherin to α-tubulin and cadherin-6 to α-tubulin in control were normalized to 1.0. (D) Immunoblotting of nuclear extracts of Tara-KD and control cells for Tbx3. The amount of nuclear Tbx3 in the Tara-KD cells was higher than that in the control cells. SB203850 blocked the increase in nuclear Tbx3 in the Tara-KD cells to the control level. (E) The phosphorylation level of nuclear Tbx3 was approximately fourfold increased in the Tara-KD cells compared with control cells, and the transfection of HA-Tara or the addition of SB203580 suppressed the increased Tbx3 phosphorylation level by ∼50%. The results are expressed as means ± SD (error bars) and are representative of three independent experiments. **, P < 0.01; *, P < 0.05.
Figure 5.

Tara-KD regulates the p38MAPK/Tbx3 pathway downstream of Rac1. (A and B) Immunoblotting for phospho-p38MAPK in Tara-KD cells in the presence or absence of SB203580 (A) or ITX3 (B). The phosphorylation level of p38MAPK was markedly up-regulated in Tara-KD cells compared with control cells. SB203580, a p38MAPK phosphorylation inhibitor, dose dependently decreased the phosphorylation level of p38, as did ITX3, an inhibitor of the Rac1-GEF activity of Trio RhoGEF. The bottom panels show the quantification of the immunoblotting data (as shown in Fig. S4 C). The results are expressed as means ± SD (error bars) and are representative of three independent experiments. **, P < 0.01; *, P < 0.05. Densitometric quantification of Western blot bands was performed using Photoshop 7.0 (Adobe). As to the “Relative Intensity,” the ratio of intensities of P-p38 to p38 in control was normalized to 1.0. (C) Immunoblotting for E-cadherin and cadherin-6 in Tara-KD cells. The Tara-KD–induced decrease of E-cadherin and increase of cahderin-6 were dose dependently inhibited by the presence of SB203580. The bottom panels show the quantification of the immunoblotting data (Fig. S4 C). The basically same results were obtained with another inhibitor of p38, SB202190. The results are expressed as means ± SD (error bars) and are representative of three independent experiments, in which antigens were immunoblotted in the same membranes. **, P < 0.01; *, P < 0.05. Densitometric quantification of Western blot bands was performed using Photoshop 7.0 (Adobe). As to the “Relative Intensity,” the ratio of intensities of E-cadherin to α-tubulin and cadherin-6 to α-tubulin in control were normalized to 1.0. (D) Immunoblotting of nuclear extracts of Tara-KD and control cells for Tbx3. The amount of nuclear Tbx3 in the Tara-KD cells was higher than that in the control cells. SB203850 blocked the increase in nuclear Tbx3 in the Tara-KD cells to the control level. (E) The phosphorylation level of nuclear Tbx3 was approximately fourfold increased in the Tara-KD cells compared with control cells, and the transfection of HA-Tara or the addition of SB203580 suppressed the increased Tbx3 phosphorylation level by ∼50%. The results are expressed as means ± SD (error bars) and are representative of three independent experiments. **, P < 0.01; *, P < 0.05.

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